This is a reissue of U.S. Pat. No. 5,770,213 issued on Jun. 23, 1998 from application Ser. No. 08/210,394 filed on May 5, 1994.
The present invention relates to P5 outer membrane protein of the Haemophilus influenzae bacterial strain and antibodies directed to P5 protein. The invention also relates to a method of isolating P5 protein and a vaccine composition for use in the treatment of Haemophilus influenzae infection.
Haemophilus influenzae strains are divided into two groups, typable and nontypable. Strains which possess a known capsule are typed by a serological reaction of the capsule with reference antisera. Currently, types a-f have been identified as typable. Strains which do not possess a capsule and fail to react with any of the reference antisera are nontypable.
Nontypable Haemophilus influenzae (NTHI) infections are implicated in several disease states including otitis media, sinusitis, and chronic pulmonary obstructive disease. Haemophilus influenzae type b (Hib) is a major cause of meningitis and other invasive infections in children under the age of four years. Antibodies directed against the capsular polysaccharide of the organism are bactericidal, opsonic in vitro and protective in experimental animals and humans. As used herein, opsonic is defined as preparation of the surface of microorganisms so that they can be more readily taken-up by phagocytes. While safe and effective vaccines for the prevention of Haemophilus influenzae type B disease have been produced, the vaccines are all based on producing antibodies to the polysaccharide capsule which is exterior to the cell wall in the bacteria. NTHi strains of Haemophilus influenzae strains by definition lack a capsule. Therefore, antibodies to capsule will not be effective at preventing NTHi infections.
It is of interest to characterize outer membrane proteins of Haemophilus influenzae bacteria and assess their vaccine potential. Munson et al., (J. Clin. Invest, 72:677-684 (1983)) reported the purification and characterization of P2, one of the major outer membrane proteins of Haemophilus influenzae strains. The researchers found that P2 protein is present in high concentrations, is easily purified, and induces protective antibody in rabbits. P5 protein is thought to be associated with the outer membrane protein layer and was previously extracted by solubilization with sodium dedecyl sulfate (SDS) and organic solvent fractionation. (Coulton et al. Can. J. Microbiology. 20:280-287 (1983)). It has been suggested, however, that the use of SDS may denature proteins in certain circumstances.
Munson and Granoff (American Society of Microbiology (p. 544. (1985)), have reported the partial characterization of P5 and P6 proteins. The results indicated that while P6 cell wall complex elicited antibody in rabbits which had protective activity in the infant rat model, P5 did not yield antiserum which was protective in infants  infant rats nor did it atisera revert wth  its antisera react with surface of bacteria by immunofluorescence which had immunofluorescence activity in vitro. Based on these findings those skilled in the art concluded that P5 was not a vaccine candidate for the prevention of disease caused by Haemophilus influenzae type b.
It is an object of the present invention, therefore, to provide essentially pure P5 outer membrane protein of Haemophilus influenzae bacteria, antibodies directed to P5 protein and a vaccine composition for use in the treatment of Haemophilus influenzae infective strains.
It is a further object of the present invention to provide a method of purifying P5 protein from the outer membrane of Haemophilus influenzae bacteria and which yields protein which can be used to produce active antibodies. Thus, P5 can be used to produce a vaccine for NTHi and type b strains of Haemophilus influenzae. The invention may be more fully understood by reference to the following drawings and detailed description.